Glutenin genes and their uses

ABSTRACT

The present invention is directed to methods of improving flour by altering glutenin content in seeds of plants, particularly wheat. The invention relates to methods of introducing a recombinant construct comprising a glutenin gene into a parental plant.

This application is a division of application Ser. No. 08/586,331, filedJan. 16, 1996, now U.S. Pat. No. 5,650,558.

FIELD OF THE INVENTION

The present invention relates generally to plant molecular biology. Inparticular, it relates to nucleic acids and methods for improvingglutenin content of plants.

BACKGROUND OF THE INVENTION

The glutenins, which include both high molecular weight (HMW) gluteninsubunits and low molecular weight (LMW) glutenin subunits, comprise aneconomically important class of wheat seed storage proteins. Theapparent molecular weights of the individual HMW glutenin polypeptidesor subunits range from 90 to 200 kDa. These subunits crosslink bydisulfide bonds among themselves and with LMW glutenin polypeptides toform polymers exceeding one million daltons in molecular weight. HMWglutenins constitute 8-10%, while LMW glutenins constitute 15-20% of thetotal endosperm protein. Both HMW and LMW glutenin proteins playimportant functional roles in determining the end-uses of wheat flour.

In wheat, HMW glutenins are encoded at the Glu-1 loci on the long armsof the group 1 chromosomes. Each locus consists of two separate genes,encoding an x-type and a y-type subunit, respectively. These pairs havenever been confirmed to be separated by recombination. This has madedetermination of their separate contributions to bread dough propertiesdifficult to assess by genetic correlation studies. For a review of thegenetics and biochemistry of glutenin polypeptides, see, Shewry et al.,J. Cereal Sci. 15:105-120 (1992).

Both the quantity and identity of specific HMW glutenin allelescontribute to the differences in bread-making quality of variouscultivars. For instance, deletion of glutenin genes results in adecrease in the overall levels of HMW glutenins, which results indecreases in bread-making quality (see, e.g., Lawrence et al. J. CerealSci 7:109-112 (1988)).

The effects of overproducing HMW glutenin on protein accumulation andbaking quality has not been assessed because such lines of wheat havenot been found among natural populations. In addition, direct alterationof the glutenin subunits that form the polymers is not possible usingstandard breeding methods. Thus, the art lacks reproducible andefficient methods of producing lines with altered glutenin contents. Thepresent invention addresses these and other needs.

SUMMARY OF THE INVENTION

The present invention provides methods of increasing glutenin in theendosperm of wheat plants. The methods and plants of the inventiontherefore are useful in providing flour and dough having improvedend-use properties. The methods comprise introducing into a parentalwheat plant a recombinant expression cassette comprising a nucleic acidencoding a glutenin polypeptide and selecting progeny wheat plant havingincreased glutenin content in the endosperm of mature seed. The glutenincontent of the progeny is preferably at least about 15% greater than theglutenin of the parental wheat plant.

Any method of introducing the expression cassette into the parentalplant can be used. Particle bombardment is a convenient method forproducing transgenic plants. Once the expression cassette is stablyintegrated into the genome, standard sexual crosses can be used tointroduce the expression cassette into desired lines.

In some embodiments, the nucleic acid introduced into the plant encodesa chimeric glutenin polypeptide. For instance, the chimeric polypeptidemay comprise sequences from an x-type glutenin polypeptide and a y-typeglutenin polypeptide. Exemplary genes for this purpose include theGlu-D1-1b gene and the Glu-D1-2b gene.

The expression cassette may further comprise a seed-specific promoter todirect expression of the introduced nucleic acid to the endosperm. Aconvenient promoter for this purpose is the promoter from the Glu-D1-2bgene.

Any wheat cultivar can be used as the parental line in the presentinvention. An exemplary cultivar is Bobwhite.

The invention also provides wheat plants comprising a recombinantexpression cassette comprising a nucleic acid encoding a gluteninpolypeptide. The plants of the invention have a glutenin content in theendosperm of a mature seed at least about 15% greater than the glutenincontent in the endosperm of a mature seed from a parental wheat plant.The percentage of total endosperm protein which is glutenin will usuallydepend upon the glutenin content of the parental line. Usually, HMWglutenins account for at least about 15% of the total protein in theendosperm of the mature seed from plants of the invention.

The invention further provides recombinant constructs comprising a wheatglutenin gene promoter of about 400 to about 2800 nucleotides operablylinked to a heterologous polynucleotide sequence. These constructs areparticularly useful in directing expression of the heterologous sequenceto seeds of transgenic plants. An exemplary wheat glutenin gene fromwhich the promoters of the invention can be derived is Glu-D1-2b.

Definitions

The term “plant” includes whole plants, plant organs (e.g., leaves,stems, roots, flowers, etc.), seeds and plant cells and progeny of same.The class of plants which can be used in the methods of the invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants.

A “heterologous sequence” is one that originates from a foreign species,or, if from the same species, is substantially modified from itsoriginal form. For example, a promoter operably linked to a heterologousstructural gene is from a species different from that from which thestructural gene was derived, or, if from the same species, one or bothare substantially modified from their original form.

A “glutenin polypeptide” is a gene product of a glutenin gene orglutenin polynucleotide sequence. A glutenin polypeptide can be either aLMW glutenin or a HMW glutenin. A glutenin polypeptide contains cysteineresidues by which disulfide bonds are formed with other gluteninpolypeptides to form polymers. The composition and size of the repeatregion is also important to polymer formation.

A “chimeric glutenin polypeptide” is glutenin gene product thatcomprises a modified amino acid sequence. Modifications, as explained indetail below, can be in the form of substitutions, deletions, oradditions of single amino acids or groups of amino acids. Thus, chimericglutenin polypeptides can be hybrid glutenin polypeptides comprisingsequences from two or more different subunits or may be polypeptides inwhich single amino acid modifications are made.

In the case where an inserted polynucleotide sequence is transcribed andtranslated to produce a functional glutenin polypeptide, one of skillwill recognize that because of codon degeneracy, a number ofpolynucleotide sequences will encode the same polypeptide. Thesevariants are specifically covered by the terms “glutenin gene” or“glutenin polynucleotide sequence”. In addition, the terms specificallyinclude those full length sequences substantially identical (determinedas described below) with a glutenin gene sequence and that encodeproteins that retain the function of the glutenin polypeptide. Thus, inthe case of wheat glutenin genes disclosed here, the term includesvariant polynucleotide sequences which have substantial identity withthe sequences disclosed here and which encode glutenin polypeptidescapable of crosslinking by disulfide bonds with other gluteninpolypeptides to form glutenin polymers.

Two polynucleotides or polypeptides are said to be “identical” if thesequence of nucleotides or amino acid residues, respectively, in the twosequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe complementary sequence is identical to all or a portion of areference polynucleotide sequence.

Sequence comparisons between two (or more) polynucleotides orpolypeptides are typically performed by comparing sequences of the twosequences over a segment or “comparison window” to identify and comparelocal regions of sequence similarity. Optimal alignment of sequences forcomparison may be conducted by the local homology algorithm of Smith andWaterman Adv. Appl. Math. 2:482 (1981), by the homology alignmentalgorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by thesearch for similarity method of Pearson and Lipman Proc. Natl. Acad.Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr.,Madison, Wis., or by inspection.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 60% sequenceidentity, preferably at least 80%, more preferably at least 90% and mostpreferably at least 95%, compared to a reference sequence using theprograms described above using standard parameters. One of skill willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like. Substantial identity of amino acidsequences for these purposes normally means sequence identity of atleast 40%, preferably at least 60%, more preferably at least 90%, andmost preferably at least 95%. Polypeptides which are “substantiallysimilar” share sequences as noted above except that residue positionswhich are not identical may differ by conservative amino acid changes.Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Stringent conditions are sequence dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. to about 20° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. However,nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This may occur, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the domains of y-type and x-type HMWglutenins. The C- and N-terminal domains are shaded, the locations ofcysteine residues are designated by S.

FIG. 2A is a diagram showing the structure of a hybrid gluteninpolypeptide of the invention.

FIG. 2B is a diagram of the recombinant plasmid of the invention whichencodes the polypeptide shown in FIG. 2A.

FIG. 3 is photograph of a gel stained with Coomassie blue showing therelative mobility of a hybrid HMW glutenin polypeptide of the invention(marked by the arrow).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to plant glutenin genes, in particular wheatglutenin genes. Nucleic acid sequences from glutenin genes can be usedto modify glutenin content in transgenic plants, in particular wheatplants. In addition, the invention provides new seed-specific promotersuseful for directing expression of desired genes in a number of plants.

Generally, the nomenclature and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art. Standard techniques are used for cloning, DNA andRNA isolation, amplification and purification. Generally enzymaticreactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. These techniques and various other techniques aregenerally performed according to Sambrook et al., Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989).

Glutenin Polypeptides and Genes

The HMW glutenins are composed primarily of a central domain of 45-90repeating two or three simple motifs, comprising chiefly glutamine andproline. This unusual primary structure results in a rod-like secondarystructure as assessed by circular dichroism and SEM. Cysteines in the C-and N-terminal domains of the molecules are known to be critical for theformation of intermolecular disulfide bonds (FIG. 1). The quaternaryinteractions of these proteins are of interest because the disulfidecrosslinks among HMW and LMW subunits is thought to be integral to theelastic properties of dough.

Any isolated glutenin gene can be used in the present invention. Theparticular polynucleotide sequence used is not a critical feature of theinvention, so long as the desired alteration in glutenin content isachieved. As noted above, HMW glutenins (both x-types and y-types) areencoded at the Glu-1 locus. In hexaploid wheat the group 1 chromosomesare designated 1A, 1B and 1D. Studies of the frequency of alleles ateach of the loci indicate the presence of at least three alleles atGlu-A1, 11 alleles at Glu-B1, and six alleles at Glu-D1. The geneproducts of each allele are designated by noting the chromosome,followed by its classification as x or y, and a number (see, MacRitchie,Advances in Food and Nutrition Research 36:1-87 (1992)).

Wheat HMW glutenin genes have been cloned and described in theliterature. For instance, a complete set of HMW glutenin genes have beenisolated and sequenced from the hard red winter wheat Cheyenne (Andersonet al. Wheat Genetics Symposium Proc. pp 699-704 (Bath Press, Cambridge,UK, 1988), including two, Dx5 and Dy10, with the highest correlationwith good flour quality (Anderson et al. Nuc. Acids. Res. 17:461-462(1989)). Other particularly useful HMW glutenins include Ax and Bxalleles.

The isolation of other glutenin genes may be accomplished by a number oftechniques. For instance, oligonucleotide probes based on the sequencesdisclosed in the prior art can be used to isolate the desired gene froma cDNA or genomic DNA library. To construct genomic libraries, largesegments of genomic DNA are generated by random fragmentation, e.g.using restriction endonucleases, and are ligated with vector DNA to formconcatemers that can be packaged into the appropriate vector. To preparea cDNA library, mRNA is isolated from endosperm and a cDNA library whichcontains the glutenin gene transcript is prepared from the mRNA.

Alternatively, the nucleic acids of interest can be amplified fromnucleic acid samples using amplification techniques. For instance,polymerase chain reaction (PCR) technology to amplify the sequences ofthe glutenin and related genes directly from genomic DNA, from cDNA,from genomic libraries or cDNA libraries. PCR and other in vitroamplification methods may also be useful, for example, to clone nucleicacid sequences that code for proteins to be expressed, to make nucleicacids to use as probes for detecting the presence of the desired mRNA insamples, for nucleic acid sequencing, or for other purposes. For ageneral overview of PCR see PCR Protocols: A Guide to Methods andApplications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.,Academic Press, San Diego (1990)).

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers et al.,Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams etal., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments maythen be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

Isolated sequences prepared as described herein can then be used tomodify glutenin gene expression and therefore glutenin content inplants. One of skill will recognize that the nucleic acid encoding afunctional glutenin protein need not have a sequence identical to theexemplified genes disclosed here. Thus, genes encoding chimeric gluteninpolypeptides can be used in the present invention.

As noted above, glutenin polypeptides, like other proteins, havedifferent domains which perform different functions. Thus, the gluteningene sequences need not be full length, so long as the desiredfunctional domains of the protein is expressed. Chimeric gluteninpolypeptides can be readily designed utilizing various recombinant DNAtechniques well known to those skilled in the art. For example, thechains can vary from the naturally occurring sequence at the primarystructure level by amino acid substitutions, additions, deletions, andthe like. In particular, cysteine residues may be added, deleted ormoved within the polypeptide to achieve a modified glutenin polypeptidewith desired properties. Chimeric polypeptides may also be produced byfusing coding sequences from two or more glutenin genes. All of thesemodifications can be used in a number of combinations to produce thefinal modified protein chain.

Preparation of Recombinant Constructs

To use isolated glutenin sequences in modifying glutenin content inplants, recombinant DNA vectors suitable for transformation of plantcells are prepared. A DNA sequence coding for the desired gluteninpolypeptide, for example a cDNA or a genomic sequence encoding a fulllength protein, is conveniently used to construct a recombinantexpression cassette which can be introduced into the desired plant. Anexpression cassette will typically comprise the glutenin polynucleotidesequence operably linked to a promoter sequence and othertranscriptional and translational initiation regulatory sequences whichwill direct the transcription of the sequence from the glutenin gene inthe intended tissues (e.g., endosperm) of the transformed plant.

For example, a constitutive plant promoter fragment may be employedwhich will direct expression of the glutenin in all tissues of a plant.Such promoters are active under most environmental conditions and statesof development or cell differentiation. Examples of constitutivepromoters include the cauliflower mosaic virus (CaMV) 35S transcriptioninitiation region, the 1′- or 2′-promoter derived from T-DNA ofAgrobacterium tumafaciens, and other transcription initiation regionsfrom various plant genes known to those of skill.

Alternatively, the plant promoter may be under environmental control.

Such promoters are referred to here as “inducible” promoters. Examplesof environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light.

Typically, the promoters used in the constructs of the invention will be“tissue-specific” and are under developmental control such that thedesired gene is expressed only in certain tissues, such as leaves,roots, fruit, seeds, or flowers. Promoters that direct expression inseeds, particularly the endosperm are particularly preferred. Examplesof such promoters include the promoter from genes encoding seed storageproteins, such as napin, cruciferin, phaseolin, and the like (see, U.S.Pat. No. 5,420,034). Other promoters suitable for expressing gluteningenes in cereals include promoters from genes encoding gliadins, cerealprolamines (e.g., zein, hordein, secalin, and avenin) and starchbiosynthetic enzymes.

The endogenous promoters from glutenin genes are particularly useful fordirecting expression of glutenin genes to the seed, particularly theendosperm. These seed-specific promoters can also be used to directexpression of heterologous structural genes. Thus, the promoters can beused in recombinant expression cassettes to drive expression of any genewhose expression in seeds is desirable. Examples include genes encodingproteins useful in increasing the nutritional value of seeds (e.g.,genes encoding proteins involved in lipid, protein, and carbohydrate orstarch biosynthesis). Other genes include those encodingpharmaceutically useful compounds, and genes encoding plant resistanceproducts to combat fungal or other infections of the seed.

The glutenin promoters can also be used to initiate transcription ofmRNA molecules to inhibit expression of an endogenous endosperm gene.Exemplary genes whose expression could be inhibited include genesencoding glutenins, enzymes involved in carbohydrate or lipid synthesis,secalins, and the like. Means for inhibiting gene expression in plantsusing recombinant DNA techniques are well known. For instance, antisensetechnology can be conveniently used. see, e.g., Sheehy et al., Proc.Nat. Acad. Sci. USA, 85:8805-8809 (1988), and Hiatt et al., U.S. Pat.No. 4,801,340. Catalytic RNA molecules or ribozymes can also be used toinhibit expression of endosperm-specific genes. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al. Nature,334:585-591 (1988). Introduction of nucleic acid configured in the senseorientation has also been shown to be an effective means by which toblock the transcription of target genes. For an example of the use ofsense suppression to modulate expression of endogenous genes see, Napoliet al., The Plant Cell 2:279-289 (1990), and U.S. Pat. Nos. 5,034,323,5,231,020, and 5,283,184.

The glutenin promoters of the invention are typically at least about 400base pairs in length, and often at least about 800 or about 1000 basepairs. The length of the promoters is typically less than about 3500base pairs, usually less than about 2800 base pairs and often less thanabout 2000 base pairs in length. The length of the promoters is countedupstream from the translation start codon of the native gene. One ofskill will recognize that use of the “about” to refer to lengths ofnucleic acid fragments is meant to include fragments of various lengthsthat do not vary significantly from the lengths recited here and stillmaintain the functions of the claimed promoters (i.e., seed-specificgene expression).

To identify glutenin promoters, the 5′ portions of a genomic gluteningene clone is analyzed for sequences characteristic of promotersequences. For instance, promoter sequence elements include the TATA boxconsensus sequence (TATAAT), which is usually 20 to 30 base pairsupstream of the transcription start site. In plants, further upstreamfrom the TATA box, at positions −80 to −100, there is typically apromoter element with a series of adenines surrounding the trinucleotideG (or T) N G. J. Messing et al., in Genetic Engineering in Plants, pp.221-227 (Kosage, Meredith and Hollaender, eds. 1983).

In preparing expression vectors of the invention, sequences other thanthe promoter and the structural gene of interest are also preferablyused. If proper polypeptide expression is desired, a polyadenylationregion at the 3′-end of the glutenin coding region should be included.The polyadenylation region can be derived from the natural gene, from avariety of other plant genes, or from T-DNA.

The vector comprising the sequences from a glutenin gene will typicallycomprise a marker gene which confers a selectable phenotype on plantcells. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosluforon, or phosphinothricin (the active ingredient inbialaphos and Basta).

Preparation of Transgenic Plants

The DNA constructs described above may be introduced into the genome ofthe desired plant host by a variety of conventional techniques.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, for example, Weising et al., Ann. Rev. Genet. 22:421-477 (1988).

The DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as biollistic methods, electroporation,PEG poration, and microinjection of plant cell protoplasts orembryogenic callus. Alternatively, the DNA constructs may be combinedwith suitable T-DNA flanking regions and introduced using anAgrobacterium tumefaciens or A. rhizogenes vector.

Particle bombardment techniques are described in Klein et al., Nature327:70-73 (1987). A particularly preferred method of transforming wheatand other cereals is the bombardment of calli derived from immatureembryos as described by Weeks et al. Plant Physiol. 102:1077-1084(1993).

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described in Paszkowski etal. Embo J. 3:2717-2722 (1984). Electroporation techniques are describedin Fromm et al. Proc. Nat. Acad. Sci. USA 82:5824 (1985).

Agrobacterium tumefaciens-meditated transformation techniques are alsowell described in the scientific literature. See, for example Horsch etal. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci.USA 80:4803 (1983). Although Agrobacterium is useful primarily indicots, certain monocots can be transformed by Agrobacterium. Forinstance, Agrobacterium transformation of rice is described by Hiei etal. i Plant J. 6:271-282 (1994).

The present invention is particularly useful in wheat and other cereals.A number of methods of transforming cereals have been described in theliterature. For instance, transformation of rice is described byToriyama et al. Bio/Technology 6:1072-1074 (1988), Zhang et al. Theor.Appl. Gen. 76:835-840 (1988), and Shimamoto et al. Nature 338:274-276(1989). Transgenic maize regenerants have been described by Fromm etal., Bio/Technology 8:833-839 (1990) and Gordon-Kamm et al., Plant Cell2:603-618 (1990)). Similarly, oats (Sommers et al., Bio/Technology10:1589-1594 (1992)), wheat (Vasil et al., Bio/Technology 10:667-674(1992)); Weeks et al., Plant Physiol. 102:1077-1084 (1993)), sorghum(Casas et al., Proc. Natl. Acad. Sci. USA 90:11212-11216 (1993)), rice(Li et al., Plant Cell Rep. 12:250-255 (1993)), barley (Yuechun andLemaux, Plant Physiol. 104:37-48 (1994)), and rye (Castillo et al.,Bio/Technology 12:1366-1371 (1994)) have been transformed viabombardment.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe glutenin polynucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans et al., Protoplasts Isolation andCulture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee etal. Ann. Rev. of Plant Phys. 38:467-486 (1987).

The methods of the present invention are particularly useful forincorporating the glutenin polynucleotides into transformed plants inways and under circumstances which are not found naturally. Inparticular, the glutenin polypeptides may be expressed at times or inquantities which are not characteristic of natural plants.

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

The invention has use over a broad range of types of plants. Theglutenin genes are preferably expressed in cereal species commonly usedfor production of flour, e.g., wheat, rye, oats, and the like.

The seed-specific promoters from glutenin genes can be used inessentially any plant species. For instance, the promoters or genesdescribed here can be used in species from the genera Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita,Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeun,Hyoscyamus, Juglans, Lactuca, Linum, Loliun, Lycopersicon, Malus,Manihot, Majorana, Medicago, Nicotiana, Oryza, Paniewn, Pannesetum,Persea, Phaseolus, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio,Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.Plants that are particularly useful in the invention include Avena,Brassica, Glycine, Hordeum, Oryza, Phaseolus, Pisum, Secale, Sorghum,Triticum, Wgna, and Zea.

As noted above, the invention is particularly useful for improvement ofwheat cultivars. Any wheat cultivar may be improved by the methods ofthe invention. Exemplary lines include hexaploid lines such as Anza,Shasta, Yecoro Rojo, Siouxland, Freedom, Tam107, and Tam200.

The effect of the modification of glutenin gene expression can bemeasured by detection of increases or decreases in desired gluteninprotein levels using, for instance, gel electrophoresis, as describedbelow. Quantification of HMW glutenin content can be carried out bySDS/PAGE densitometry, as described below.

Other methods for quantifying glutenin content include sonication offlour dispersions in SDS buffer to solubilize glutenin and otherrelatively insoluble proteins (see, Singh and MacRitchie in WheatEnd-Use Properties: Wheat and Flour Characterization for SpecificEnd-Uses H. Salovaara, ed., pp 321-326 (University of Helsinki,Helsinki, 1989). This method is useful in solubilizing at least 95 % oftotal flour protein. Quantification of the glutenin fraction can then beperformed using size exclusion high performance liquid chromatography asdescribed by Batey et al., Cereal Chem. 68:207-209 (1991). Othersuitable methods include reverse phase HPLC and capillaryelectrophoresis.

The plants of the invention have increased in glutenin content in theendosperm of mature seeds as compared to the parental lines from whichthey are derived. The increase is usually at least about 15% as comparedto the parental line. More usually, the increase is at least about 20%,preferably at least about 35%, and in more preferred embodiments atleast about 45 %. Any of the methods for quantifying glutenins in theendosperm described above can be used to determine the percent increasein glutenin content. A wheat seed typically reaches maturity about 6 toabout 10 weeks after anthesis.

Another measure of increased glutenin content in transgenic plants ofthe invention is glutenin content as a percent of total protein of amature seed or flour derived from the mature seed. Different wheatcultivars have different glutenin contents as measured using standardmethods. Thus, the final glutenin content of the plants of the inventionwill depend upon the parental line. In the case where HMW glutenin genesare used, the plants of the invention will typically have HMW glutenincontents of at least about 15%, usually at least about 18%, andpreferably at least about 20% as measured using standard methods such asthose described in detail below.

Analysis of the rheological properties of flour derived from thetransgenic plants of the invention can be carried out according tostandard physical dough-testing instruments widely used to measure flourand dough quality (see, e.g., MacRitchie, Advances in Food and NutritionResearch 36:1-87 (1992)). Such methods include, for instance, use ofextensographs to measure tensile strength. Two of the main parametersmeasured are maximum resistance (R_(max)) and extensibility (Ext). Othermethods include mixographs and bake-test loaf volume (MacRitchie andGras Cereal Chem. 50:292-302 (1973) as well as SDS-sedimentation tests,amelographs, and cookie spread methods.

The following Examples are offered by way of illustration, notlimitation.

EXAMPLE 1

This example demonstrates increase in glutenin content in transgenicwheat plants comprising recombinant constructs of the invention.

Materials and Methods

Construction of the Hybrid HMW Glutenin Expression Plasmid

Two HMW glutenin subunits, Dx5 and Dy10, encoded by the Glu-D1-1b andGlu-D1-2b genes, respectively, have been most often associated withsuperior dough strength by genetic correlation studies. A bacterialexpression vector comprising a fusion of coding regions of the Dy10 andDx5 genes is described by Shani et al., Plant Physiol. 98:433-441 (1992). The hybrid HMW glutenin polypeptide encoded by this gene is diagrammedin FIG. 2A.

In this example, the Dy10:Dx5 hybrid coding region was reunited with 5′and 3′ flanking sequences from the native Dy10 and Dx5 genes. Theconstruction of a fusion at the HindIII site located at base pairs429-435 and 385-391 after the A of the start codons of the Glu-D1-2b(Genbank Accession No. X12929) and Glu-D1-1b (Genbank Accession No.X12928) genes (Anderson et al. Nuc. Acids Res. 17:461-462 (1989)) andits insertion into the bacterial expression vector pet3A to formpet-3a-10/5 is described by Shani et al., supra.

Thus, the native Dy10 gene provides the promoter, transcription startsite, the 5′ transcribed untranslated region and the first 145 codonsincluding the 21 amino acid signal peptide. The native Dx5 gene providesits codons 130 to 848 followed by two translation stop codons, thetranscription termination and poly(A) addition signals. Because aminoacids 138 through 147 are shared by the Dy10 and Dx5 glutenin subunits,the central region of repeating amino acid motifs of the hybrid gluteninpolypeptide (thickest boxes in FIG. 2A) is identical to that of Dx5. Theconfiguration of the cysteines (S) and thus of potential disulfide bondsis chimeric in nature: the N-terminal cysteines are of the y-type andthe single C-terminal cysteine is typical of x-type subunits.

In order to reconstitute the promoter region, transcription start andtermination sites, and any other regulatory signals needed forexpression in wheat, three fragments were isolated and combined in asingle ligation reaction with the Bluescript KS⁻ (Stratagene) vector cutwith EcoRI and BamHI: 1) the 715 bp StuI/DrdI fragment that contains thejunction region in pET-3a-10/5; 2) the 2800 bp fragment from a clone ofthe native Dy10 gene beginning at the EcoRI site 5′ to the gene andending at the StuI site 74 bp after the A in the start codon; and 3) the3800 bp fragment from a clone of the native Dx5 gene that starts at theDrdI site 744 bp after the A in the start codon and ends at a BamHI sitein the vector just outside the EcoRI site in the 3′ flanking region. Aplasmid with the correct structure was identified by restrictionanalysis and named pGlu10H5 (FIG. 2B). This plasmid was deposited underterms of the Budapest Treaty in the Agricultural Research CultureCollection (NRRL), 1815 North University Street, Peoria, Ill., on Jan.12, 1996 and has been assigned accession number NRRL B-21517.

The plasmid UBI:BAR which comprises the maize ubiquitin promoteroperably linked to the bar gene encoding the enzyme PAT, whichinactivates phosphinothricin was prepared as described Cornejo et al.Plant Mol. Biol. 23:567-581 (1993).

Both plasmids were prepared by the alkaline lysis method using a Qiagenkit for the final purification. The DNAs were stored in TE buffer at aconcentration of 1 mg/ml.

Wheat Transformation

12.5 ug of each DNA (an approximately 2:1 molar ratio of UBI:BAR topGlu10H5) was coated onto gold particles for bombardment into immaturewheat embryos of cultivar Bobwhite essentially as described by Weeks etal. Plant Physiol. 102:1077-1084 (1993) except that the embryos wereincubated on callus induction media containing mannitol for 4 hoursbefore and 20 hours after bombardment. Transformants were selected onthe basis of their resistance to 1 mg/L bialaphos at the callus andgreen shoot stages of regeneration and to 3 mg/L bialaphos at therooting stage of regeneration as described by Weeks et al., supra.

Protein Analysis

Protein extracts from immature wheat endosperm were prepared by grindingthe tissue or cells in SDS-PAGE sample loading buffer [66 mM Tris pH6.8, 3% (w/v) SDS, 0.05% (w/v) Bromphenol blue, 2% (v/v)β-mercaptoethanol]. Protein extracts from mature dry wheat seeds wereprepared by dissolving flour in sample buffer. SDS-PAGE was according tostandard procedures. The separation gels for detection and quantitationof the hybrid subunit were 10% (w/v) acrylamide 0.05% (w/v)bis-acrylamide. The gels were run at 40 mamps constant current until 20minutes after the bromphenol blue dye front had run off the gel (about1000 Volt-hours).

The gels were stained by the Neuhoff method with a colloidal suspensionof Coomassie blue. Protein standards for molecular weight calibrationwere purchased from Pharmacia and consisted of Phosphorylase b (94 kDa)Bovine Serum Albumin (67 kDa), Ovalbumin (43 kDa), Carbonic Anhydrase(30 kDa), Soybean Trypsin Inhibitor (20.1 kDa) and α-Lactalbumin (14.4kDa). Stained gels were scanned using an Alpha Innotech densitometer.

DNA Analysis

Southern analyses of genomic DNA was as described in Weeks et al.,supra, except that 10 ug of DNA were loaded into each slot of a 0.6%agarose gel. The probe consisted of the 1578 bp PvuII fragment from thecoding region of Dx5 (bracketed in FIG. 2B).

Results

Forty-five independent callus pieces were selected from 4980 embryosbombarded in 10 different experiments; each yielded one to five plants.The T₀ primary regenerated plants and the lines derived from them aredesignated with an unique number following the bombardment number, e.g.,plant 126 regenerated from bombardment 7 is 7-126. Plants fromtwenty-six of these independent line gave rise to progeny embryos thatwere able to germinate in the presence of 3 mg/L bialaphos when excisedfrom immature seeds three to four weeks after anthesis.

The immature endosperm tissue corresponding to each excised embryo wasextracted and screened by SDS-PAGE for the presence of the hybridglutenin. This SDS-PAGE analysis showed that the migration of the hybridHMW glutenin subunit is clearly distinguishable from the native HMWsubunits under these gel conditions.

Although the lanes did not contain the same amounts of total protein, itwas evident that there is variation in the expression levels of thehybrid subunit relative to those of the native HMW subunits in the sameseed. Of the twenty-six resistant lines that were derived fromco-bombardment experiments including pGlu10OH5, sixteen (60%) linesshowed expression of the hybrid subunit in the T₁ generation.

Expression Stability

At each successive generation, 24 or 25 immature embryos 3-4 weeks afteranthesis were tested for their ability to germinate on bialaphos. Plantswere classified as homozygous if all 25 of their progeny embryosexhibited resistance. The levels of transgene expression were assessedin successive generations. In most cases, expression was stable. Anapparent increase in expression between the T1 and T2 generations insome plants was explained by the increase in average gene dosage betweenthe heterozygous and homozygous progeny of the T0 plant and T1generation, respectively. Since the endosperm is triplicate, individualselfed progeny of the To heterozygote could have 0, 1, 2 or 3 copies andthe T1 lane contains an average of 8 of these individuals.

In four of the 15 wheat lines analyzed, expression of the hybrid HMWglutenin has not remained at the same levels, but rather has declined ashomozygous resistant lines were selected in the T₂ or the T₃generations. The decline could be due either to hybrid HMW glutenintransgene inactivation or to loss of unlinked pGlu10H5 transgene(s)copies via segregation.

Cross-linking Ability of the Hybrid Subunit

As noted above, the hybrid HMW glutenin contains a unique configurationof cysteines, y-type at the N-terminus and x-type at the C-terminus. Thehybrid HMW glutenin behaved exactly like the native subunits in terms ofits solubility in SDS buffers with and without a reducing agent, whenexamined by SDS-PAGE. The native and hybrid HMW glutenins were partiallyextracted from both the Bobwhite and the transgenic flours by SDS aloneand were present in lower molecular weight polymers. The majority of thehybrid glutenin, however, was insoluble in SDS buffer and could only beextracted when a reducing agent was added. Thus, the configuration ofcysteines in the hybrid HMW glutenin does not preclude the formation ofeither intermolecular disulfide bonds or high molecular weight polymers.

Relative Expression Levels

FIG. 3 shows SDS-PAGE analysis of protein extracts from eight differenttransgenic lines (Lanes 1-8) and Bobwhite (C). Each sample was preparedfrom flour of a mixture of eight mature seeds of a single homozygous T₂or T₃ plant. Under these gel conditions, the native By9 and Dy10subunits are not well resolved and run as a closely spaced doublet ofapparent molecular weight 94 kDa. Likewise the two isoforms of the Dx5subunit are not well-resolved and migrate as a broad band. The Bx7 bandis well-separated from other proteins and the darkest of the HMWsubunits in intensity. In all the lines except the one shown in Lane 3,the hybrid HMW subunit is present in at least as high a quantity as thenative Ax2* (largest) HMW subunit. Thus even a single copy of the hybridtransgene (Lane 1) can support significant expression levels, comparableto those of the native HMW subunits.

In one of the lines (shown in lane 2), synthesis of native HMW gluteninsynthesis has declined relative to the other proteins. The extract shownthere exhibits reduced synthesis of all the native HMW glutenin comparedto the lower molecular weight seed proteins in the same lane.

In order to estimate the levels of the hybrid HMW glutenin relative tothose of the native HMW glutenin and other seed proteins, the gel wasscanned by densitometry. All the wheat prolamines stay on the gel.However, since proteins of molecular weights less than 33 kDa are runoff the gel under the conditions used to separate the hybrid HMWglutenin from the nearby Dx5 subunit band, only relative quantitiescould be assessed. The results of these comparisons are shown in Table Ialong with the number of intact transgene copies estimated fromrestriction enzyme digests of genomic DNA (see below). The hybrid HMWglutenin band constitutes more than 20% of the total HMW glutenins inall the lines except that in lane 3. Thus the transgene has made asignificant contribution to the HMW glutenin composition in all thetransgenic lines.

In the case where endogenous HMW glutenin accumulation is reduced (Lane2), the hybrid makes up 62% of the total complement of HMW glutenin. Inmost lines, the sum of the HMW glutenin accumulation is elevated 29 to45% relative to the HMW glutenin content of Bobwhite (all sums arenormalized to the remainder of the protein their respective lanes). Thusthis quantitative analysis suggests that addition of HMW-glutenin genecopies raises the levels of HMW glutenin accumulation in mature seeds.

DNA Analysis

DNA was isolated from the leaves of transgenic plants of each line andassayed for the presence of transgenes by genomic Southern analysis, asdescribed above. Hybridizing bands known to correspond to the transgeneswere scanned and their densities compared. The results are shown inTable I. The number ranges from 1 in line 11-291 to about 6 in 14-23(Table I). This range is much broader than that in total HMW gluteninlevels or even hybrid HMW glutenin levels. However the most highlyexpressing line had one of the highest copy numbers (Line 4) and thepoorest expression was in a single copy line (Line 3).

TABLE I Transgenic HMW-glutenin gene activity in individual wheat lines.Transgene Total HMW subunit Gene Copy No. Line Expression as % of totalEstimate 1 23% 135% 1 2 62%  70% 5-6 3 15% 145% 1 4 42% 145% 4-5 5 23%140% 2 6 24% 129% 2 7 22% 135% 3-4 8 20% 143% 3-4

Discussion

The experiments reported here show that levels of HMW glutenins can beincreased by addition of gene copies. The behavior of the hybrid subunitis indistinguishable from that of the native subunits. The proteinaccumulates in the endosperm tissue over the course of seed developmentand is cross-linked by disulfide bonds into large polymers that cannotbe extracted by SDS without addition of reducing agents.

Expression of the hybrid glutenin is under the control of the nativeDy10 5′ and Dx5 3′ flanking sequences. These sequences were sufficientto achieve high levels of the modified seed storage protein. Even asingle gene copy supported expression levels comparable to those of thenative HMW glutenin genes. Thus, these sequences serve as a source ofeffective regulatory sequences suitable for expression of other proteinsin transgenic wheat and potentially other cereal endosperm.

EXAMPLE 2

This example describes the transformation of wheat using a recombinantconstruct encoding Dx5 and Dy10.

The recombinant expression vectors were prepared as generally asdescribed above except that the complete Dx5 or Dy10 gene, including 5′and 3′ regulatory sequences, were used. The constructs were thenintroduced into immature wheat embryos by particle bombardment asdescribed above. Analysis of T₀ plants and their progeny is performed asdescribed above.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference for all purposes.

What is claimed is:
 1. Wheat flour derived from a plant comprising arecombinant expression cassette comprising a nucleic acid encoding aglutenin polypeptide and a seed-specific promoter, the plant having aglutenin content in the endosperm of a mature seed at least about 30%different than the glutenin content in the endosperm of a mature seedfrom a parental wheat plant.